Simulated blood culture samples were done using selected multidrug resistant clinical isolates that were stored in −80°C. These isolates were thawed and cultured overnight on blood agar plates at 37°C. Resulting pure colonies were suspended in 0.9% NaCl until a turbidity of 0.5 McFarland (1.5 × 108 CFU/mL) before being diluted to a final 15,000 CFU/mL, from which 70 μL (1,000 CFU) was mixed with 5 mL of sterile human blood from healthy donors (Transfusion Medicine, Karolinska University Hospital, Huddinge) and inoculated into a BacT/Alert-FA Plus bottle (bioMérieux, Marcy-l'Étoile, France). The inoculated bottles were then incubated in the BacT/Alert 3D system (bioMérieux, Marcy-l'Étoile, France) and were removed after signaling positive. The bacterial suspension was also cultured on two blood agar plates as an inoculation CFU control, and CFU controls showed that the inoculum had 13 ± 5 CFU/μL. In addition, negative control bottles inoculated with the human blood without bacteria were incubated in the same way, and these were automatically discarded by the BacT/Alert 3D system (bioMérieux, Marcy-l'Étoile, France) after 5 days of incubation without detected microbial growth.
Bact alert 3d system
Manufactured by bioMérieux
Sourced in France, United States
The BacT/ALERT 3D system is a fully automated, continuous-monitoring blood culture system designed for the detection of aerobic and anaerobic microorganisms in patient samples. The system utilizes colorimetric sensors to monitor for changes in carbon dioxide production, which indicates microbial growth.
Automatically generated - may contain errors
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71 protocols using bact alert 3d system
1
Simulated Blood Culture Assay
2
Evaluating Antimicrobial Susceptibility of Bacteremia
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We collected 15 residual specimens of a positive blood culture bottle (BacT/Alert® FA or BacT/Alert® FN) from adult patients with bacteremia at Toyama University Hospital after the detection of bacterial growth with the BacT/ALERT 3D system (bioMerieux, Inc., Mercy-l’Etoile, France). All specimens were confirmed to be monomicrobial with Gram staining before use, because conventional antimicrobial susceptibility (and even identification) had not been completed at the time of enrollment. However, they were eventually confirmed to be monomicrobial using solid media at the Clinical Laboratory Center (certified ISO15189) at Toyama University Hospital. The MIC values determined by the conventional method, as reference values, were determined with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, IL, USA) in the clinical laboratory.
3
Monitoring Neonatal Bacterial Colonization
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Blood cultures were processed for the diagnosis of bacteremia with automated microbial detection systems BacT/Alert 3D system (bioMérieux SA, Marcy l’Etoile, France). To determine the evolution of HAIs in neonatal patients and colonized babies, all newborns admitted were routinely screened for bacterial colonization and received a nasopharynx and rectum examination on their arrival in the unit and on a weekly basis following the admission. Rectal and cavum swabs collected from patients and surfaces were inoculated on Drigalski agar (bioMérieux SA, Marcy l’Etoile, France). All inoculated samples were incubated at 36°C for 48 h. The isolates recovered were routinely identified using MALDI-TOF MS.
4
Blood culture identification protocol
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A single culture bottle inoculated with a blood sample was incubated in an automated BacT/ALERT 3D system (Biomerieux, France) for a maximum of 5 days. Blood cultures flagged as positive were Gram-stained and subcultured on MacConkey, chocolate, and blood agar plates (Oxide, UK) following standard microbiological techniques. Bacterial isolates were identified based on colony morphology, Gram reaction and biochemical reaction. Yeasts on Gram stained smears from positive blood cultures were identified based on morphology; no attempt was made to identify the fungal species. Staphylococcus aureus was differentiated from coagulase-negative staphylococci (CoNS) based on slide and tube coagulase test methods [27 ]. An isolate was considered a blood culture contaminant in instances when CoNS, viridans streptococci, Micrococcus species, Bacillus species, and Corynebacterium species were detected [14 (link), 28 (link)].
5
Fungal Isolate Identification and Antifungal Susceptibility Testing
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Blood cultures were performed using the BacT/alert 3D system (bioMérieux, Lyon, France). All fungal isolates from blood cultures were identified with VITEKTM2 (bioMérieux, Lyon, France) using CHROMagarTM Candida broth (Becton Dickinson Japan, Tokyo, Japan).
The MIC measurement followed the methodology of the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and used yeast-like fungi DP-Eiken (Tokyo, Japan), with higher values adopted when MICs differed in the same isolate. The MIC measurement ranges were as follows: fluconazole 0.12–64 µg/mL, itraconazole and voriconazole 0.015–8 µg/mL, amphotericin B and caspofungin 0.03–16 µg/mL, and micafungin 0.015–16 µg/mL. Posaconazole and anidulafungin were not approved in Japan during the study period. MIC50 and MIC90 were calculated for each species. MIC50 and MIC90 are defined as the concentrations of each antifungal agent necessary to inhibit 50% and 90% of the isolates, respectively. MIC > 0.06 µg/mL was used as a criterion for the low susceptibility of micafungin for non-parapsilosis Candida species. MIC > 0.06 µg/mL was set with reference to the resistance norm of C. glabrata in CLSI M60 1st Edition (Performance Standards for Antifungal Susceptibility Testing of Yeasts) [23 ].
The MIC measurement followed the methodology of the Clinical and Laboratory Standards Institute (CLSI) M27-A3 and used yeast-like fungi DP-Eiken (Tokyo, Japan), with higher values adopted when MICs differed in the same isolate. The MIC measurement ranges were as follows: fluconazole 0.12–64 µg/mL, itraconazole and voriconazole 0.015–8 µg/mL, amphotericin B and caspofungin 0.03–16 µg/mL, and micafungin 0.015–16 µg/mL. Posaconazole and anidulafungin were not approved in Japan during the study period. MIC50 and MIC90 were calculated for each species. MIC50 and MIC90 are defined as the concentrations of each antifungal agent necessary to inhibit 50% and 90% of the isolates, respectively. MIC > 0.06 µg/mL was used as a criterion for the low susceptibility of micafungin for non-parapsilosis Candida species. MIC > 0.06 µg/mL was set with reference to the resistance norm of C. glabrata in CLSI M60 1st Edition (Performance Standards for Antifungal Susceptibility Testing of Yeasts) [23 ].
6
Evaluation of Direct VITEK MS and VITEK2 AST for Positive Blood Cultures
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This study was approved by Emory IRB and was conducted in Grady Memorial Hospital, a 960-bed, inner city teaching hospital, during the period from 8/1/12 to 5/30/13. One hundred positive blood cultures from 100 different patients were included in the study. As there were no polymicrobic bottles seen during the study period, only monomicrobic isolates from positive blood bottles were studied.
Blood samples collected and inoculated in BacT/ALERT® anaerobic (SN) and standard aerobic (SA) non-charcoal based blood culture bottles, were incubated in the BacT/ ALERT 3D system (bioMérieux, Durham, NC). When a signal-positive bottle was detected, aliquot was taken from positive bottles for Gram stain and subculture on solid media. Isolates grown from culture media were used for ID and full panel AST by using conventional methods such as MicroScan (Siemens, West Sacramento, CA) and VITEK® 2 (bioMérieux). In parallel, aliquot taken from positive blood culture bottles were processed using the LFM for direct ID by VITEK MS and full panel AST by VITEK2.
Blood samples collected and inoculated in BacT/ALERT® anaerobic (SN) and standard aerobic (SA) non-charcoal based blood culture bottles, were incubated in the BacT
7
Microbial Identification in Clinical Specimens
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Microbial cultures were used to identify the organism present in biological specimens derived from different sites (respiratory tract, urinary tract, abdomen, and others) according to clinical suspicion by standardized microbiological cultural assays procedures.
Blood cultures were performed in patients with clinical symptoms of bloodstream infections prior the administration of antimicrobial therapy. For each patient, two bottles sets were used for each septic episode; approximately 10 mL of blood was inoculated in the aerobic and anaerobic bottle (BACT/ALERT Culture Media, Biomerieux, Marcy-l’Etoile, France); the bottles were entered in the BACT/ALERT 3D system for the incubation and measure of the color change in response to shift in pH as a result of rising of carbon dioxide (CO2) levels produced by microorganisms. In positive samples, bacteria and yeasts were identified on the Vitek 2 system (Biomerieux, Marcy-l’Etoile, France), according to manufacturer directions.
Blood cultures were performed in patients with clinical symptoms of bloodstream infections prior the administration of antimicrobial therapy. For each patient, two bottles sets were used for each septic episode; approximately 10 mL of blood was inoculated in the aerobic and anaerobic bottle (BACT/ALERT Culture Media, Biomerieux, Marcy-l’Etoile, France); the bottles were entered in the BACT/ALERT 3D system for the incubation and measure of the color change in response to shift in pH as a result of rising of carbon dioxide (CO2) levels produced by microorganisms. In positive samples, bacteria and yeasts were identified on the Vitek 2 system (Biomerieux, Marcy-l’Etoile, France), according to manufacturer directions.
8
Isolation and Cultivation of E. coli Strains
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The E. coli reference strain and the ESBL E. coli were grown under atmospheric conditions at 37 °C in cation adjusted Mueller-Hinton broth with TES (CAMHBT) overnight. The blood culture E. coli strain was isolated from a blood sample from a patient with bacteremia which were collected in standard aerobe non-charcoal BacT/ALERT FA blood culture bottles (bioMérieux, Marcy l’Etoile, France), and handled according to the manufacturer’s instructions. Blood culture bottles were incubated in the BacT/ALERT 3D system (bioMérieux, Marcy-l’Etoile, France) at Aarhus University hospital (Aarhus, Denmark). The blood culture sample was prepared by centrifugation at 200 × g for 5 min (Sigma 3–18 k, 12171 rotor, Buch & Holm, Herlev, Denmark) to remove human blood cells, followed by resuspension of the bacterial pellet in CAMHBT.
9
Automated Bacteraemia Identification Workflow
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Blood samples from patients (8–10 mL/patient) with suspected bacteraemia were collected in standard aerobe non-charcoal BacT/ALERT FA blood culture bottles (bioMérieux, Marcy-l’Etoile, France) and handled according to the manufacturer’s instructions. Blood culture bottles were incubated in the BacT/ALERT 3D system (bioMérieux, Marcy-l’Etoile, France) at Aarhus University Hospital (Aarhus, Denmark). Blood cultures detected as positive by the BacT/ALERT 3D system were Gram stained and examined for bacterial morphology by light microscopy. All blood cultures used in this study were recognised as positive during the night and not processed until 8 o’clock the following morning. Species identification of isolates by the clinical laboratory was provided after the completion of oCelloScope AST measurements. Consequently, only preliminary phenotypic characterisation including Gram staining and examination of cellular morphology by light microscopy was provided prior to oCelloScope AST.
10
Blood Culture and Pathogen Identification Protocol
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Upon preliminary confirmation of BSI, whole blood samples were obtained for blood culture and molecular diagnosis. Two sets of blood cultures were collected from each patient according to routine clinical practice; each set consisted of one aerobic bottle and one anaerobic bottle. The blood cultures were incubated at 37°C in a BacT/ALERT® 3D System (BioMérieux). When a positive signal was obtained, Gram staining was performed, followed by subculturing on a Columbia blood agar plate at 37°C with 5% CO2. Following overnight incubation, the pathogens were further identified via matrix‐assisted laser desorption‐ionization time‐of‐flight mass spectrometry (VITEK® MS system; BioMérieux). The positive control bacteria comprised A. baumannii ATCC 19606 along with five clinical isolates and K. pneumoniae CMCC 46117 along with 33 clinical isolates. A total of 131 other clinical isolates commonly found in BSIs were used as negative controls (Table 2 ).
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